Method of treating graft versus host disease

ABSTRACT

A method for preventing the development of or treating GvHD complications in a mammalian patient which comprises administering to the mammal a population of cells enriched for STRO-1bright cells and/or progeny thereof and/or soluble factors derived therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/055,221, filed Feb. 26, 2016, which is a continuation of U.S.application Ser. No. 13/808,093, filed Mar. 14, 2013, now U.S. Pat. No.9,301,978, issued May 4, 2016, which is a § 371 national stage of PCTInternational Application No. PCT/AU2011/000840, filed Jul. 4, 2011,claiming the benefit of U.S. Provisional Application No. 61/398,950,filed Jul. 2, 2010, the contents of each of which are herebyincorporated by reference into the application.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“180914_81809-AAA-PCT-US_Sequence_Listing_CAE.txt”, which is 7.27kilobytes in size, and which was created Sep. 14, 2018 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Sep. 14, 2018 aspart of this application.

FIELD

This invention relates to methods for enhancing the engraftment ofhematopoietic progenitor cells, enhancing bone marrow transplantationand preventing or reducing graft versus host disease. In one embodimentthe invention relates to preventing or alleviating the complicationsfollowing allogeneic bone marrow transplantation, namely graft versushost disease in mammalian patients, especially in human patients.

BACKGROUND

Bone marrow transplantation is indicated following a process whichdestroys bone marrow. For example, following intensive systemicradiation or chemotherapy, bone marrow is the first target to fail.Metastatic cancers are commonly treated with very intensivechemotherapy, which is intended to destroy the cancer, but alsoeffectively destroys the bone marrow. This induces a need for bonemarrow transplantation. Alleviation of any but the most acutelife-threatening conditions involving bone marrow disorders with bonemarrow transplantation is, however, generally regarded as too risky,because of the likelihood of the onset of graft versus host disease(GvHD).

GvHD is an immunological disorder that is the major factor that limitsthe success and availability of allogeneic bone marrow or stem celltransplantation. GvHD is a systemic inflammatory reaction which causeschronic illness and may lead to death of the host mammal. At present,allogeneic transplants invariably run a severe risk of associated GvHD,even where the donor has a high degree of histocompatibility with thehost.

GvHD is caused by donor T-cells reacting against systemicallydistributed incompatible host antigens, causing powerful inflammation.In GvHD, mature donor T-cells that recognize differences between donorand host become systemically activated. Current methods to prevent andtreat GvHD involve administration of drugs such as cyclosporin-A andcorticosteroids. These have serious side effects, must be given forprolonged periods of time, and are expensive to administer and tomonitor. Attempts have also been made to use T-cell depletion to preventGvHD, but this requires sophisticated and expensive facilities andexpertise. Too great a degree of T-cell depletion leads to seriousproblems of failure of engraftment of bone marrow stem cells, failure ofhematopoietic reconstitution, infections, or relapse. More limitedT-cell depletion leaves behind cells that are still competent toinitiate GvHD. As a result, current methods of treating GvHD are onlysuccessful in limited donor and host combinations, so that many patientscannot be offered potentially life-saving treatment.

Mesenchymal stem cells (MSC) exhibit a potent immunosuppressive activitywhich has been successfully exploited in the clinical setting to treatgraft-versus-host disease (GvHD), an otherwise lethal complication ofbone marrow transplantation. Because of the limited characterization,MSC preparations are quite heterogenous and this limits the magnitude oftheir immunosuppression and therefore the clinical benefit.

SUMMARY

In work leading up to the present invention, the inventors comparedmesenchymal stem cell and STRO-1^(bright) multipotential cellpreparations in terms of their effect on GvHD. Surprisingly, theStro-1^(bright) multipotential cell preparation was vastly superior tothe mesenchymal stem cell preparation in ameliorating GvHD.

Accordingly the present invention provides a method for alleviating thedevelopment of GvHD complications in a mammalian patient which comprisesadministering to the patient a population of cells enriched forSTRO-1^(bright) cells and/or progeny thereof and/or soluble factorsderived therefrom.

In one embodiment the mammalian patient is undergoing or about toundergo a bone marrow transplant.

In another embodiment the present invention provides a method for ofalleviating the development of GvHD complications in a mammalian patientcaused by bone marrow transplantation which comprises administering tothe patient (a) precursors of bone marrow lineage cells, and (b) apopulation of cells enriched for STRO-1^(bright) cells and/or progenythereof and/or soluble factors derived therefrom, wherein the populationof cells enriched for STRO-1^(bright) cells and/or progeny thereofand/or soluble factors derived therefrom is/are administered in anamount effective to reduce the severity of GvHD in the patient.

In one embodiment, the graft-versus-host disease is a result of a T cellimmune response. In one example, the T cells are from a donor and theantigen is from the recipient. For example, the T cells may be presentin a transplant. In another embodiment, the T cells are from therecipient and the antigen is from the donor.

In another embodiment of this method the STRO-1^(bright) cells and/orprogeny cells thereof and/or soluble factors derived therefrom aregenetically engineered to express a molecule to block co-stimulation ofT-cells.

The STRO-1^(bright) cells may be autogeneic or allogeneic. In oneembodiment, the STRO-1^(bright) cells are allogeneic.

In another embodiment of this method, the STRO-1^(bright) cells and/orprogeny cells thereof have been expanded in culture prior toadministration or to obtaining the soluble factors.

Exemplary dosages of the cells include between 0.1×10⁶ to 5×10⁶STRO-1^(bright) cells and/or progeny thereof. For example, the methodcomprises administering between 0.3×10⁶ to 2×10⁶ STRO-1^(bright) cellsand/or progeny thereof.

One form of the method involves administering a low dose ofSTRO-1^(bright) cells and/or progeny thereof. Such a low dose is, forexample, between 0.1×10⁵ and 0.5×10⁶ STRO-1^(bright) cells and/orprogeny thereof, such as about 0.3×10⁶ STRO-1^(bright) cells and/orprogeny thereof.

The present disclosure also contemplates numerous administrations of thecells and/or soluble factors. For example, such a method can involveadministering the cells and monitoring the subject to determine when oneor more symptoms of GvHD occurs or recurs and administering a furtherdose of the cells and/or soluble factors. Suitable methods for assessingsymptoms of GvHD will be apparent to the skilled artisan and/ordescribed herein.

In one example, the population enriched for STRO-1^(bright) cells and/orprogeny thereof and/or soluble factors derived therefrom areadministered once weekly or less often, such as, once every four weeksor less often.

In another embodiment, the population of cells enriched forSTRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom is administered systemically. For example, thepopulation of cells enriched for Stro-1^(bri) cells and/or progeny cellsthereof and/or soluble factors derived therefrom may be administeredintravenously, intra-arterially, intramuscularly, subcutaneously, intoan aorta, into an atrium or ventricle of the heart or into a bloodvessel connected to an organ, e.g., an abdominal aorta, a superiormesenteric artery, a pancreaticoduodenal artery or a splenic artery.

In another embodiment the methods of the invention further compriseadministering an immunosuppressive agent. The immunosuppressive agentmay be administered for a time sufficient to permit said transplantedcells to be functional. In one example, the immunosuppressive agent iscyclosporine. The cyclosporine may be administered at a dosage of from 5to 40 mg/kg body wt.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Co-expression of TNAP (STRO-3) and the Mesenchymal PrecursorCell Marker, STRO-1^(bright) by Adult Human BMMNC. Dual-colorimmunofluorescence and flow cytometry was performed by incubation ofSTRO-1 MACS-selected BMMNC and indirectly labelled with a goatanti-murine IgM antibody coupled to FITC (x axis), and STRO-3 mAb(murine IgG1) indirectly labelled with a goat anti-murine IgG coupled toPE (y axis). The dot plot histogram represents 5×10⁴ events collected aslistmode data. The vertical and horizontal lines were set to thereactivity levels of <1.0% mean fluorescence obtained with theisotype-matched control antibodies, 1B5 (IgG) and 1A6.12 (IgM) treatedunder the same conditions. The results demonstrate that a minorpopulation of STRO-1^(bright) cells co-expressed TNAP (upper rightquadrant) while the remaining STRO-1+ cells failed to react with theSTRO-3 mAb.

FIG. 2. Gene expression profile of STRO-1^(bright) or STRO-1^(dim)progeny of cultured and expanded STRO-1^(bright) MPC. Single cellsuspensions of ex vivo expanded bone marrow MPC were prepared bytrypsin/EDTA treatment. Cells were stained with the STRO-1 antibodywhich was subsequently revealed by incubation with goat-anti murineIgM-fluorescein isothiocyanate. Total cellular RNA was prepared frompurified populations of STRO-1^(dim) or STRO-1^(bright) expressingcells, following fluorescence activated cell sorting (A). Using RNAzolBextraction method, and standard procedures, total RNA was isolated fromeach subpopulation and used as a template for cDNA synthesis. Theexpression of various transcripts was assessed by PCR amplification,using a standard protocol as described previously (Gronthos et al. JCell Sci. 116:1827-1835, 2003). Primers sets used in this study areshown in Table 2. Following amplification, each reaction mixture wasanalysed by 1.5% agarose gel electrophoresis, and visualised by ethidiumbromide staining (B). Relative gene expression for each cell marker wasassessed with reference to the expression of the house-keeping gene,GAPDH, using ImageQant software (C).

FIG. 3. STRO-1^(bright) progeny of cultured and expanded STRO-1⁺ MPCexpress high levels of SDF-1, STRO-1^(dim) progeny do not. (A)MACS-isolated preparations of STRO-1⁺ BMMNCs were partitioned intodifferent STRO-1 subsets according to the regions, STRO-1^(bright) andSTRO-1^(dim/dull) using FACS. Total RNA was prepared from each STRO-1subpopulation and used to construct a STRO-1^(bright) subtractionhybridization library (B-C). Replicate nitrocellulose filters, whichhave been blotted with representative PCR products amplified frombacterial clones transformed with STRO-1^(bright) subtracted cDNA. Thefilters were then probed with either [³²P] deoxycytidine triphosphate(dCTP)-labeled STRO-1^(bright) (B) or STRO-1^(dim/dull) (C) subtractedcDNA. The arrows indicate differential expression of 1 clone containinga cDNA fragment corresponding to human SDF-1. (D) Reverse transcriptase(RT)-PCR analysis demonstrating the relative expression of SDF-1 andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in totalRNA prepared from freshly MACS/FACS-isolated BMMNC STRO-1 populationsprior to culture. bp indicates base pair.

FIG. 4. Comparative efficiency of STRO-1 negative MSC (preparation A)and STRO-1^(bright) MPC (preparation B) for inhibition of T cellproliferation. PBMC were stimulated with CD3/CD28 coated beads for 4days in the absence or presence of preparations A or B. T cellproliferation was measured by ³H-Tdr incorporation as counts per minute(cpm).

FIG. 5. Comparative efficiency of STRO-1 negative MSC (preparation A)and STRO-1^(bright) MPC (preparation B) for inhibition of T cellproliferation. PBMC were stimulated with CD3/CD28 coated beads for 4days in the presence of different concentrations of preparations A or B.T cell proliferation in the various cultures were was measured by ³H-Tdrincorporation and reported as percentage of the control T cellproliferation in which PBMC were stimulated in the absence of MSC.

FIG. 6. Comparative effects of STRO-1 negative MSC (preparation A) andSTRO-1^(bright) MPC (preparation B) on GvHD. T-cell depleted bone marrowmononuclear cells (BMMC) (5×10⁶) and splenocytes (30×10⁶) from B10.D2(H2d) donors were injected intravenously into lethally irradiated (750cGy) BALB/c (H2d) recipient mice. After 4 weeks, recipient mice wereinjected with 1×10⁶ MSC (preparation A1) or MPC (preparation B1) permouse or received no further treatment (Control). Eight mice per groupwere injected. Mice were assessed at weekly interval. Time refer tonumber of weeks.

FIG. 7. Comparative effects of STRO-1 negative MSC (preparation A) andSTRO-1^(bright) MPC (preparation B) on GvHD. T-cell depleted bone marrowmononuclear cells (BMMC) (5×10⁶) and splenocytes (30×10⁶) from B10.D2(H2d) donors were injected intravenously into lethally irradiated (750cGy) BALB/c (H2d) recipient mice. After 4 weeks, recipient mice wereinjected with 1 or 2×10⁶ cells of preparations A or B (A1, A2, B1, B2)or received no further treatment (Ctr). Eight mice per group wereinjected.

FIG. 8. Comparative effects of high-dose STRO-1 negative MSC(preparation A) and STRO-1^(bright) MPC (preparation B) on GvHD. T-celldepleted bone marrow mononuclear cells (BMMC) (5×10⁶) and splenocytes(30×10⁶) from B10.D2 (H2d) donors were injected intravenously intolethally irradiated (750 cGy) BALB/c (H2d) recipient mice. After 4weeks, recipient mice were injected with 2×10⁶ cells of preparations Aor B (A2, B2) or received no further treatment (Control 2). Eight miceper group were injected but 4 and 3 mice died soon after the injectionin groups B2 and A2, respectively.

FIG. 9. Comparative effects of low-dose STRO-1 negative MSC (preparationA) and STRO-1^(bright) MPC (preparation B) on GvHD. T-cell depleted bonemarrow mononuclear cells (BMMC) (5×10⁶) and splenocytes (30×10⁶) fromB10.D2 (H2d) donors were injected intravenously into lethally irradiated(750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, recipient micewere injected with 0.3×10⁶ cells of preparations A or B (A0.3,B0.3) orreceived no further treatment (Control 2). Six mice per group wereinjected. The graph reports the GvHD score per each mouse.

FIG. 10 Comparative effects of low-dose STRO-1 negative MSC (preparationA) and STRO-1^(bright) MPC (preparation B) on GvHD. T-cell depleted bonemarrow mononuclear cells (BMMC) (5×10⁶) and splenocytes (30×10⁶) fromB10.D2 (H2d) donors were injected intravenously into lethally irradiated(750 cGy) BALB/c (H2d) recipient mice. After 4 weeks, recipient micewere injected with 0.3×10⁶ cells of preparations A or B (A0.3, B0.3) orreceived no further treatment (Control 2). Six mice per group wereinjected. The graph reports the average GvHD score in each group.

DETAILED DESCRIPTION

Results presented herein show that a population of cells enriched forSTRO-1^(bright) cells was unexpectedly vastly superior to a STRO-1negative mesenchymal stem cell preparation in ameliorating GvHD.

Accordingly the present invention provides a method for alleviating thedevelopment of GvHD complications in a mammalian patient which comprisesadministering comprising administering to the patient a population ofcells enriched for STRO-1^(bright) cells and/or progeny thereof and/orsoluble factors derived therefrom.

For example, the invention provides a method for of alleviating thedevelopment of GvHD complications in a mammalian patient caused by bonemarrow transplantation which comprises administering to the patient (a)precursors of bone marrow lineage cells, and (b) a population of cellsenriched for STRO-1^(bright) cells and/or progeny thereof and/or solublefactors derived therefrom, wherein the population of cells enriched forStro-1^(bright) cells and/or progeny thereof and/or soluble factorsderived therefrom is/are administered in an amount effective to reducethe severity of GvHD in the patient.

As used herein, the term “soluble factors” shall be taken to mean anymolecule, e.g., protein, peptide, glycoprotein, glycopeptide,lipoprotein, lipopeptide, carbohydrate, etc. produced by STRO-1^(bright)cells and/or progeny thereof that are water soluble. Such solublefactors may be intracellular and/or secreted by a cell. Such solublefactors may be a complex mixture (e.g., supernatant) and/or a fractionthereof and/or may be a purified factor. In one embodiment of thepresent invention soluble factors are or are contained withinsupernatant. Accordingly, any embodiment herein directed toadministration of one or more soluble factors shall be taken to applymutatis mutandis to the administration of supernatant.

The methods of the invention may involve administration of population ofcells enriched for STRO-1^(bright) cells and/or progeny cells thereofalone, and/or soluble factors derived therefrom. The methods of theinvention may also involve administration of progeny cells alone, orsoluble factors derived from the progeny cells. The methods of theinvention may also involve administration of a mixed population ofSTRO-1^(bright) cells and progeny cells thereof, or soluble factors froma mixed culture of STRO-1^(bright) cells and progeny cells thereof.

A preferred application of this invention is to humans, however, it isexpected that the invention is also applicable to animals, and thesemight include agricultural animals such as cows, sheep, pigs and thelike, domestic animals such as dogs, cats, laboratory animals such asmice, rats, hamsters and rabbits or animals that might be used for sportsuch as horses.

Thus, STRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom can be used to condition a recipient's immunesystem to donor or foreign bone marrow cells by administering to therecipient, prior to, or at the same time as transplantation of the donorcells, STRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom in an amount effective to reduce or eliminatean immune response against the transplant by the recipient's T cells.The STRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom affect the T cells of the recipient such thatthe T cell response is reduced or eliminated when presented with donoror foreign tissue.

Thus, in the context of bone marrow (hematopoietic stem cell)transplantation, attack of the host by the graft can be reduced oreliminated. Donor marrow can be pretreated with recipientSTRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom prior to implant of the bone marrow orperipheral blood stem cells into the recipient. In a preferredembodiment, the donor marrow is first exposed to recipient tissue/cellsand then treated with STRO-1^(bright) cells and/or progeny cells thereofand/or soluble factors derived therefrom. Although not being limitedthereto, it is believed that the initial contact with recipient tissueor cells functions to activate the T cells in the marrow. Subsequenttreatment with the STRO-1^(bright) cells and/or progeny cells thereofand/or soluble factors derived therefrom inhibits or eliminates furtheractivation of the T cells in the marrow, thereby reducing or eliminatingan adverse affect by the donor tissue, i.e. the therapy reduces oreliminates graft versus host response.

In a further embodiment, a transplant recipient suffering from graftversus host disease may be treated to reduce or eliminate the severitythereof by administering to such recipient STRO-1^(bright) cells and/orprogeny cells thereof and/or soluble factors derived therefromautologous or allogeneic to the donor, which allogeneic cells can beSTRO-1^(bright) cells and/or progeny cells thereof autologous to therecipient or third party STRO-1^(bright) cells and/or progeny cellsthereof, in an amount effective to reduce or eliminate a graft rejectionof the host. The STRO-1^(bright) cells and/or progeny cells thereofand/or soluble factors derived therefrom inhibit or suppress theactivated T cells in the donor tissue from mounting an immune responseagainst the recipient, thereby reducing or eliminating a graft versushost response.

The recipient's STRO-1^(bright) cells and/or progeny cells thereofand/or soluble factors derived therefrom may be obtained from therecipient prior to the transplantation and may be stored and/orculture-expanded to provide a reserve of STRO-1^(bright) cells and/orprogeny cells thereof and/or soluble factors derived therefrom insufficient amounts for treating an ongoing graft attack against host.

It is further contemplated that only a single treatment with theSTRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom of the present invention may be required,eliminating the need for chronic immunosuppressive drug therapy.Alternatively, multiple administrations of Stro-1^(bri) cells and/orprogeny cells thereof and/or soluble factors derived therefrom may beemployed.

The dosage of the STRO-1^(bright) cells and/or progeny cells thereofand/or soluble factors derived therefrom varies within wide limits andwill, of course be fitted to the individual requirements in eachparticular case. In general, in the case of parenteral administration,it is customary to administer from about 0.01 to about 5 million cellsper kilogram of recipient body weight. The number of cells used willdepend on the weight and condition of the recipient, the number of orfrequency of administrations, and other variables known to those ofskill in the art

The cells can be suspended in an appropriate diluent, at a concentrationof from about 0.01 to about 5×10⁶ cells/ml. One form of the methodinvolves administering a low dose of STRO-1^(bright) cells and/orprogeny thereof. Such a low dose is, for example, between 0.1×10⁵ and0.5×10⁶ STRO-1^(bright) cells and/or progeny thereof, such as about0.3×10⁶ STRO-1^(bright) cells and/or progeny thereof.

Suitable excipients for injection solutions are those that arebiologically and physiologically compatible with the cells and with therecipient, such as buffered saline solution or other suitableexcipients. The composition for administration is preferably formulated,produced and stored according to standard methods complying with propersterility and stability.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

STRO-1^(bright) Cells or Progeny Cells, and Supernatant or One or MoreSoluble Factors Derived Therefrom

STRO-1^(bright) cells are cells found in bone marrow, blood, dental pulpcells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver,heart, retina, brain, hair follicles, intestine, lung, lymph node,thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum;and are typically capable of differentiating into germ lines such asmesoderm and/or endoderm and/or ectoderm. Thus, STRO-1^(bright) cellsare capable of differentiating into a large number of cell typesincluding, but not limited to, adipose, osseous, cartilaginous, elastic,muscular, and fibrous connective tissues. The specificlineage-commitment and differentiation pathway which these cells enterdepends upon various influences from mechanical influences and/orendogenous bioactive factors, such as growth factors, cytokines, and/orlocal microenvironmental conditions established by host tissues.STRO-1^(bright) cells are thus preferably non-hematopoietic progenitorcells which divide to yield daughter cells that are either stem cells orare precursor cells which in time will irreversibly differentiate toyield a phenotypic cell.

In a preferred embodiment, the STRO-1^(bright) cells are enriched from asample obtained from a subject, e.g., a subject to be treated or arelated subject or an unrelated subject (whether of the same species ordifferent). The terms ‘enriched’, ‘enrichment’ or variations thereof areused herein to describe a population of cells in which the proportion ofone particular cell type or the proportion of a number of particularcell types is increased when compared with the untreated population.

In one embodiment the STRO-1^(bright) cells are preferentially enrichedrelative to STRO-1^(dim) or STRO-1^(intermediate) cells.

In a preferred embodiment, the cells used in the present inventionexpress one or more markers individually or collectively selected fromthe group consisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺,3G5⁺ or any combination thereof.

By “individually” is meant that the invention encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the invention encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

Preferably, the STRO-1^(bright) cells are additionally one or more ofTNAP⁺, VCAM-1⁺, THY-1⁺ STRO-2⁺ and/or CD146⁺.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This terms means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labelled or is undetectableabove background levels.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker protein (forexample the antigen recognised by STRO-1) than other cells in thesample. For instance, STRO-1^(bright) cells produce a greaterfluorescent signal, when labelled with a FITC-conjugated STRO-1 antibodyas determined by fluorescence activated cell sorting (FACS) analysis,than non-bright cells (STRO-1^(dull/dim)). Preferably, “bright” cellsconstitute at least about 0.1% of the most brightly labelled bone marrowmononuclear cells contained in the starting sample. In otherembodiments, “bright” cells constitute at least about 0.1%, at leastabout 0.5%, at least about 1%, at least about 1.5%, or at least about2%, of the most brightly labelled bone marrow mononuclear cellscontained in the starting sample. In a preferred embodiment,STRO-1^(bright) cells have 2 log magnitude higher expression of STRO-1surface expression relative to “background”, namely cells that areSTRO-1⁻. By comparison, STRO-1^(dim) and/or STRO-1^(intermediate) cellshave less than 2 log magnitude higher expression of STRO-1 surfaceexpression, typically about 1 log or less than “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In aparticularly preferred embodiment, TNAP as used herein refers to amolecule which can bind the STRO-3 antibody produced by the hybridomacell line deposited with ATCC on 19 Dec. 2005 under the provisions ofthe Budapest Treaty under deposit accession number PTA-7282.

Furthermore, in a preferred embodiment, the STRO-1^(bright) cells arecapable of giving rise to clonogenic CFU-F.

It is preferred that a significant proportion of the multipotentialcells are capable of differentiation into at least two different germlines. Non-limiting examples of the lineages to which the multipotentialcells may be committed include bone precursor cells; hepatocyteprogenitors, which are multipotent for bile duct epithelial cells andhepatocytes; neural restricted cells, which can generate glial cellprecursors that progress to oligodendrocytes and astrocytes; neuronalprecursors that progress to neurons; precursors for cardiac muscle andcardiomyocytes, glucose-responsive insulin secreting pancreatic betacell lines. Other lineages include, but are not limited to,odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another embodiment, the Stro-1^(bright) cells are not capable ofgiving rise, upon culturing, to hematopoietic cells.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used. In another useful embodiment of the invention,cells of a non-human animal (or if the patient is not a human, fromanother species) are used.

The present invention also contemplates use of supernatant or solublefactors obtained or derived from STRO-1^(bright) cells and/or progenycells thereof (the latter also being referred to as expanded cells)which are produced from in vitro culture. Expanded cells of theinvention may a have a wide variety of phenotypes depending on theculture conditions (including the number and/or type of stimulatoryfactors in the culture medium), the number of passages and the like. Incertain embodiments, the progeny cells are obtained after about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10passages from the parental population. However, the progeny cells may beobtained after any number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an embodiment, progeny cells useful for the methods of the inventionare obtained by isolating TNAP⁺ STRO-1⁺ multipotential cells from bonemarrow using magnetic beads labelled with the STRO-3 antibody, and thenculture expanding the isolated cells (see Gronthos et al. Blood 85:929-940, 1995 for an example of suitable culturing conditions).

In one embodiment, such expanded cells (progeny) (preferably, at leastafter 5 passages) can be TNAP⁻, CC9⁺, HLA class I⁺, HLA class II⁻,CD14⁻, CD19⁻, CD3⁻, CD11a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one preferred embodiment, expandedcells still have the capacity to differentiate into different celltypes.

In one embodiment, an expended cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 25%, more preferably at least 50%, of the cells are CC9+.

In another embodiment, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, more preferably at least 45%, of the cells are STRO-1⁺.

In a further embodiment, the expanded cells may express one or moremarkers collectively or individually selected from the group consistingof LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-1^(bright)and CD146 or any combination of these markers.

In one embodiment, progeny cells derived from STRO-1^(bright) cells arepositive for the marker Stro-1^(dim). These cells are referred to asTissue Specific Committed Cells (TSCCs) and are more committed todifferentiation than STRO-1^(bri) cells are therefore less able torespond inductive factors. Non-limiting examples of the lineages towhich TSCCs may be committed include hepatocyte progenitors, which arepluripotent for bile duct epithelial cells and hepatocytes; neuralrestricted cells, which can generate glial cell precursors that progressto oligodendrocytes and astrocytes, and neuronal precursors thatprogress to neurons; precursors for cardiac muscle and cardiomyocytes,glucose-responsive insulin secreting pancreatic beta cell lines. Othercommitted precursor cells include but are not limited to chondrocytes,osteoblasts, odontoblast, dentin-producing and chondrocytes, andprecursor cells of the following: retinal pigment epithelial cells,fibroblasts, skin cells such as keratinocytes, dendritic cells, hairfollicle cells, renal duct epithelial cells, smooth and skeletal musclecells, testicular progenitors, vascular endothelial cells, tendon,ligament, cartilage, adipocyte, fibroblast, marrow stroma, osteoclastand haemopoietic-supportive stroma, cardiac muscle, smooth muscle,skeletal muscle, pericyte, vascular, epithelial, glial, neuronal,astrocyte and oligodendrocyte cells. Precursors include those thatspecifically can lead to connective tissue particularly includingadipose, areolar, osseous, cartilaginous, elastic and fibrous connectivetissues.

In another embodiment, the progeny cells are Multipotential ExpandedSTRO-1⁺ Multipotential cells Progeny (MEMPs) as defined and/or describedin WO 2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising MPCs from which progeny are derived maybe directly harvested from a tissue source, or alternatively it may be apopulation that has already been expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP, STRO-1^(bright), 3G5⁺, VCAM-1, THY-1,CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested STRO-1^(bright) cellsin that they are positive for the marker STRO-1^(bright) and negativefor the marker Alkaline phosphatase (ALP). In contrast, freshly isolatedStro-1^(bri) cells are positive for both STRO-1^(bright) and ALP. In apreferred embodiment of the present invention, at least 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have thephenotype STRO-1^(bri), ALP⁻. In a further preferred embodiment theMEMPS are positive for one or more of the markers Ki67, CD44 and/orCD49c/CD29, VLA-3, α3β1. In yet a further preferred embodiment the MEMPsdo not exhibit TERT activity and/or are negative for the marker CD18.

The STRO-1^(bright) cell starting population may be derived from any oneor more tissue types including bone marrow, dental pulp cells, adiposetissue and skin, or perhaps more broadly from adipose tissue, teeth,dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles,intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament,bone marrow, tendon and skeletal muscle.

It will be understood that in performing the present invention,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, preferred methodsrely upon binding a binding agent (e.g., an antibody or antigen bindingfragment thereof) to the marker concerned followed by a separation ofthose that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody-based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. The separation techniques preferably maximisethe retention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques.

It is preferred that the method for isolating STRO-1^(bright) cells, forexample, comprises a first step being a solid phase sorting steputilising for example magnetic activated cell sorting (MACS) recognisinghigh level expression of STRO-1. A second sorting step can then follow,should that be desired, to result in a higher level of precursor cellexpression. This second sorting step might involve the use of two ormore markers.

The method obtaining STRO-1^(bright) cells might also include theharvesting of a source of the cells before the first enrichment stepusing known techniques. Thus the tissue will be surgically removed.Cells comprising the source tissue will then be separated into a socalled single cells suspension. This separation may be achieved byphysical and or enzymatic means.

Once a suitable STRO-1^(bright) cell population has been obtained, itmay be cultured or expanded by any suitable means to obtain MEMPs.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used to obtain the supernatant or soluble factors. Inanother useful embodiment of the invention, cells of a non-human animal(or if the patient is not a human, from another species) are used toobtain supernatant or soluble factors.

The invention can be practised using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the invention may be performed include but are not limited tocells of chimpanzees, baboons, cynomolgus monkeys, and any other New orOld World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the invention may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which theinvention may be performed.

Cells useful for the methods of the invention may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.).

Genetically-Modified Cells

In one embodiment, the STRO-1^(bright) cells and/or progeny cellsthereof are genetically modified, e.g., to express and/or secrete aprotein of interest, e.g., a protein providing a therapeutic and/orprophylactic benefit, e.g., insulin, glucagon, somatostatin,trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreaticlipase or amylase or a polypeptide associated with or causative ofenhanced angiogenesis or a polypeptide associated with differentiationof a cell into a pancreatic cell or a vascular cell.

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present embodiment of the invention.

Preferably, the nucleic acid is provided in the form of an expressionconstruct. As used herein, the term “expression construct” refers to anucleic acid that has the ability to confer expression on a nucleic acid(e.g. a reporter gene and/or a counter-selectable reporter gene) towhich it is operably connected, in a cell. Within the context of thepresent invention, it is to be understood that an expression constructmay comprise or be a plasmid, bacteriophage, phagemid, cosmid, virussub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the invention will be apparent to the skilled artisan andare described, for example, in Ausubel et al (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable forthe method of the present invention in a mammalian cell is, for example,a vector of the pcDNA vector suite supplied by Invitrogen, a vector ofthe pCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Invitrogen Corporation, Clontech orPromega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the invention is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include, for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739(1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al,Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller andRosman BioTechniques 7:980-990, 1989; Miller, A. D. Human Gene Therapy7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc.Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 andWO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka.Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, HumanGene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169,1994; and Zhou et al. J Exp. Med. 179:1867-1875, 1994.

Additional viral vectors useful for delivering an expression constructof the invention include, for example, those derived from the pox familyof viruses, such as vaccinia virus and avian poxvirus or an alphavirusor a conjugate virus vector (e.g. that described in Fisher-Hoch et al.,Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Assaying Therapeutic/Prophylactic Potential of Cells and Soluble Factors

Methods for determining the ability of soluble factors derived fromSTRO-1^(bright) cells to treat or prevent or delay the onset orprogression of GvHD will be apparent to the skilled artisan.

For example, suitable in vitro tests for determining immunosuppressiveactivity of the soluble factors are described in Example 5 herein.

In another example, efficacy of soluble factors may be assessed in an invivo model of GvHD as described in Examples 6 and 7 herein.

It will be apparent to the skilled artisan from the foregoing that thepresent disclosure also provides a method for identifying or isolating asoluble factor for the treatment, prevention or delay of GvHD, themethod comprising:

-   (i) administering a a soluble factor to a test subject suffering    from GvHD and assessing progression of GvHD in the subject;-   (ii) comparing level of GvHD in the subject at (i) to the level GvHD    in a control subject suffering from GvHD to which the soluble factor    has not been administered, wherein reduced GvHD in the test subject    compared to the control subject indicates that the soluble factor    treats, prevents or delays GvHD.    Cellular Compositions

In one embodiment of the present invention STRO-1^(bright) cells and/orprogeny cells thereof are administered in the form of a composition.Preferably, such a composition comprises a pharmaceutically acceptablecarrier and/or excipient.

The terms “carrier” and “excipient” refer to compositions of matter thatare conventionally used in the art to facilitate the storage,administration, and/or the biological activity of an active compound(see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., MacPublishing Company (1980). A carrier may also reduce any undesirableside effects of the active compound. A suitable carrier is, for example,stable, e.g., incapable of reacting with other ingredients in thecarrier. In one example, the carrier does not produce significant localor systemic adverse effect in recipients at the dosages andconcentrations employed for treatment.

Suitable carriers for this invention include those conventionally used,e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, abuffered solution, hyaluronan and glycols are preferred liquid carriers,particularly (when isotonic) for solutions. Suitable pharmaceuticalcarriers and excipients include starch, cellulose, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesiumstearate, sodium stearate, glycerol monostearate, sodium chloride,glycerol, propylene glycol, water, ethanol, and the like.

In another example, a carrier is a media composition, e.g., in which acell is grown or suspended. Preferably, such a media composition doesnot induce any adverse effects in a subject to whom it is administered.

Preferred carriers and excipients do not adversely affect the viabilityof a cell and/or the ability of a cell to reduce, prevent or delaypancreatic dysfunction.

In one example, the carrier or excipient provides a buffering activityto maintain the cells and/or soluble factors at a suitable pH to therebyexert a biological activity, e.g., the carrier or excipient is phosphatebuffered saline (PBS). PBS represents an attractive carrier or excipientbecause it interacts with cells and factors minimally and permits rapidrelease of the cells and factors, in such a case, the composition of theinvention may be produced as a liquid for direct application to theblood stream or into a tissue or a region surrounding or adjacent to atissue, e.g., by injection.

STRO-1^(bright) cells and/or progeny cells thereof can also beincorporated or embedded within scaffolds that are recipient-compatibleand which degrade into products that are not harmful to the recipient.These scaffolds provide support and protection for cells that are to betransplanted into the recipient subjects. Natural and/or syntheticbiodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in thepractice of the invention. Preferred scaffolds include, but are notlimited to biological, degradable scaffolds. Natural biodegradablescaffolds include collagen, fibronectin, and laminin scaffolds. Suitablesynthetic material for a cell transplantation scaffold should be able tosupport extensive cell growth and cell function. Such scaffolds may alsobe resorbable. Suitable scaffolds include polyglycolic acid scaffolds,e.g., as described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima,et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr.Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold(such as Gelfoam from Upjohn Company).

The cellular compositions useful for the present invention may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentinvention include, but are not limited to, other multipotent orpluripotent cells or stem cells, or bone marrow cells. The cells ofdifferent types may be admixed with a composition of the inventionimmediately or shortly prior to administration, or they may beco-cultured together for a period of time prior to administration.

Preferably, the composition comprises an effective amount or atherapeutically or prophylactically effective amount of cells. Forexample, the composition comprises about 1×10⁵ STRO-1^(bright) cells/kgto about 1×10⁷ STRO-1^(bright) cells/kg or about 1×10⁶ STRO-1^(bright)cells/kg to about 5×10⁶ STRO-1^(bright) cells/kg. The exact amount ofcells to be administered is dependent upon a variety of factors,including the age, weight, and sex of the patient, and the extent andseverity of the pancreatic dysfunction.

In some embodiments, cells are contained within a chamber that does notpermit the cells to exit into a subject's circulation, however thatpermits factors secreted by the cells to enter the circulation. In thismanner soluble factors may be administered to a subject by permittingthe cells to secrete the factors into the subject's circulation. Such achamber may equally be implanted at a site in a subject to increaselocal levels of the soluble factors, e.g., implanted in or near atransplanted organ.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions. Accordingly, transplantation withallogeneic, or even xenogeneic, STRO-1^(bright) cells or progeny thereofmay be tolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. The cells may be encapsulated in acapsule that is permeable to nutrients and oxygen required by the celland therapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, the cells may be geneticallymodified to reduce their immunogenicity.

Compositions of Soluble Factors

In one embodiment of the present invention, STRO-1^(bright) cell-derivedand/or progeny cell-derived supernatant or soluble factors areadministered in the form of a composition, e.g., comprising a suitablecarrier and/or excipient. Preferably, the carrier or excipient does notadversely affect the biological effect of the soluble factors orsupernatant.

In one embodiment, the composition comprises a composition of matter tostabilize a soluble factor or a component of supernatant, e.g., aprotease inhibitor. Preferably, the protease inhibitor is not includedin an amount sufficient to have an adverse effect on a subject.

Compositions comprising STRO-1^(bright) cell-derived and/or progenycell-derived supernatant or soluble factors may be prepared asappropriate liquid suspensions, e.g., in culture medium or in a stablecarrier or a buffer solution, e.g., phosphate buffered saline. Suitablecarriers are described herein above. In another example, suspensionscomprising Stro-1^(bri) cell-derived and/or progeny cell-derivedsupernatant or soluble factors are oily suspensions for injection.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil; or synthetic fatty acid esters, such as ethyl oleate ortriglycerides; or liposomes. Suspensions to be used for injection mayalso contain substances which increase the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol, or dextran.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

Sterile injectable solutions can be prepared by incorporating thesupernatant or soluble factors in the required amount in an appropriatesolvent with one or a combination of ingredients described above, asrequired, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the supernatant orsoluble factors into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In accordance with an alternative aspect of theinvention, the supernatant or soluble factors may be formulated with oneor more additional compounds that enhance its solubility.

Other exemplary carriers or excipients are described, for example, inHardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the soluble factors may be administered ina time release formulation, for example in a composition which includesa slow release polymer. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PLG). Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art.

The supernatant or soluble factors may be administered in combinationwith an appropriate matrix, for instance, to provide slow release of thesoluble factors.

Modes of Administration

The STRO-1^(bright) cell-derived supernatant or soluble factors,STRO-1^(bright) cells or progeny thereof may be surgically implanted,injected, delivered (e.g., by way of a catheter or syringe), orotherwise administered directly or indirectly to the site in need ofrepair or augmentation, e.g., an organ or into the blood system of asubject.

Preferably, the STRO-1^(bright) cell-derived supernatant or solublefactors, STRO-1^(bright) cells or progeny thereof is delivered to theblood stream of a subject. For example, the STRO-1^(bright) cell-derivedsupernatant or soluble factors, STRO-1^(bright) cells or progeny thereofare delivered parenterally. Exemplary routes of parenteraladministration include, but are not limited to, intravenous,intramuscular, subcutaneous, intra-arterial, intraperitoneal,intraventricular, intracerebroventricular, intrathecal. Preferably, theSTRO-1^(bright) cell-derived supernatant or soluble factors,STRO-1^(bright) cells or progeny thereof are delivered intra-arterially,into an aorta, into an atrium or ventricle of the heart or into a bloodvessel connected to a pancreas, e.g., an abdominal aorta, a superiormesenteric artery, a pancreaticoduodenal artery or a splenic artery.

In the case of cell delivery to an atrium or ventricle of the heart, itis preferred that cells are administered to the left atrium or ventricleto avoid complications that may arise from rapid delivery of cells tothe lungs.

Preferably, the STRO-1^(bright) cell-derived supernatant or solublefactors, STRO-1^(bright) cells or progeny thereof are injected into thesite of delivery, e.g., using a syringe or through a catheter or acentral line.

Selecting an administration regimen for a therapeutic formulationdepends on several factors, including the serum or tissue turnover rateof the entity, the level of symptoms, and the immunogenicity of theentity. Preferably, an administration regimen maximizes the amount oftherapeutic compound delivered to the patient consistent with anacceptable level of side effects. Accordingly, the amount of formulationdelivered depends in part on the particular entity and the severity ofthe condition being treated.

In one embodiment, STRO-1^(bright) cell-derived supernatant or solublefactors, STRO-1^(bright) cells or progeny thereof are delivered as asingle bolus dose. Alternatively, STRO-1^(bright) cell-derivedsupernatant or soluble factors, STRO-1^(bright) cells or progeny thereofare administered by continuous infusion, or by doses at intervals of,e.g., one day, one week, or 1-7 times per week. A preferred doseprotocol is one involving the maximal dose or dose frequency that avoidssignificant undesirable side effects. A total weekly dose depends on thetype and activity of the compound being used. Determination of theappropriate dose is made by a clinician, e.g., using parameters orfactors known or suspected in the art to affect treatment or predictedto affect treatment. Generally, the dose begins with an amount somewhatless than the optimum dose and is increased by small incrementsthereafter until the desired or optimum effect is achieved relative toany negative side effects. Important diagnostic measures include thoseof symptoms of diabetes.

EXAMPLES Example 1: MSC Preparation

MSCs are generated de novo from bone marrow as described in U.S. Pat.No. 5,837,539. Approximately 80-100 ml of marrow was aspirated intosterile heparin-containing syringes and taken to the MDACC Cell TherapyLaboratory for MSC generation. The bone marrow mononuclear cells wereisolated using ficoll-hypaque and placed into two T175 flask with 50 mlper flask of MSC expansion medium which includes alpha modified MEM(αMEM) containing gentamycin, glutamine (2 mM) and 20% (v/v) fetalbovine serum (FBS) (Hyclone).

The cells were cultured for 2-3 days in 37° C., 5% CO₂ at which time thenon-adherent cells were removed; the remaining adherent cells werecontinually cultured until the cell confluence reached 70% or higher(7-10 days), and then the cells were trypsinized and replaced in sixT175 flasks with MSC expansion medium (50 ml of medium per flask). Asdescribed in Table 5 of U.S. Pat. No. 5,837,539, MSCs isolated andexpanded in this manner are STRO-1 negative.

Example 2: Immunoselection of MPCs by Selection of STRO-3+ Cells

Bone marrow (BM) is harvested from healthy normal adult volunteers(20-35 years old), in accordance with procedures approved by theInstitutional Ethics Committee of the Royal Adelaide Hospital. Briefly,40 ml of BM is aspirated from the posterior iliac crest intolithium-heparin anticoagulant-containing tubes.

BMMNC are prepared by density gradient separation using Lymphoprep™(Nycomed Pharma, Oslo, Norway) as previously described (Zannettino, A.C. et al. (1998) Blood 92: 2613-2628). Following centrifugation at 400×gfor 30 minutes at 4° C., the buffy layer is removed with a transferpipette and washed three times in “HHF”, composed of Hank's balancedsalt solution (HBSS; Life Technologies, Gaithersburg, Md.), containing5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

STRO-3⁺ (or TNAP⁺) cells were subsequently isolated by magneticactivated cell sorting as previously described (Gronthos et al. (2003)Journal of Cell Science 116: 1827-1835; Gronthos, S. and Simmons, P. J.(1995) Blood 85: 929-940). Briefly, approximately 1-3×10⁸ BMMNC areincubated in blocking buffer, consisting of 10% (v/v) normal rabbitserum in HHF for 20 minutes on ice. The cells are incubated with 200 μlof a 10 μg/ml solution of STRO-3 mAb in blocking buffer for 1 hour onice. The cells are subsequently washed twice in HHF by centrifugation at400×g. A 1/50 dilution of goat anti-mouse γ-biotin (SouthernBiotechnology Associates, Birmingham, UK) in HHF buffer is added and thecells incubated for 1 hour on ice. Cells are washed twice in MACS buffer(Ca²⁺- and Mn²⁺-free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01%sodium azide) as above and resuspended in a final volume of 0.9 ml MACSbuffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0.5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb(deposited on 19 Dec. 2005 with American Type Culture Collection (ATCC)under accession number PTA-7282—see International Publication No. WO2006/108229). After addition of a further 1 ml MACS buffer, the columnis removed from the magnet and the TNAP⁺ cells are isolated by positivepressure. An aliquot of cells from each fraction can be stained withstreptavidin-FITC and the purity assessed by flow cytometry.

Example 3: Cells Selected by STRO-3 mAb are STRO-1^(bright) Cells

Experiments were designed to confirm the potential of using STRO-3 mAbas a single reagent for isolating cells STRO-1^(bright) cells.

Given that STRO-3 (IgG1) is a different isotype to that of STRO-1 (IgM),the ability of STRO-3 to identify clonogenic CFU-F was assessed bytwo-colour FACS analysis based on its co-expression with STRO-1⁺ cellsisolated using the MACS procedure (FIG. 1). The dot plot histogramrepresents 5×10⁴ events collected as listmode data. The vertical andhorizontal lines were set to the reactivity levels of <1.0% meanfluorescence obtained with the isotype-matched control antibodies, 1B5(IgG) and 1A6.12 (IgM) treated under the same conditions. The resultsdemonstrate that a minor population of STRO-1^(bright) cellsco-expressed TNAP (upper right quadrant) while the remaining STRO-1⁺cells failed to react with the STRO-3 mAb. Cells isolated by FACS fromall four quadrants were subsequently assayed for the incidence of CFU-F(Table 1).

TABLE 1 Enrichment of human bone marrow cells by dual-colour FACSanalysis based on the co-expression of the cell surface markers STRO-1and TNAP (refer to FIG. 1). FACS sorted cells were cultured understandard clonogenic conditions in alpha MEM supplemented with 20% FCS.The data represents the mean number of day 14 colony-forming cells(CFU-F) per 10⁵ cells plated ± SE (n = 3 different bone marrowaspirates). These data suggest that human MPC are exclusively restrictedto the TNAP positive fraction of BM which co-express the STRO-1 antigenbrightly. Frequency of Enrichment Bone Marrow Fraction CFU-F/10⁵ Cells(Fold Increase) Unfractionated BMMNC  11.0 ± 2.2 1.0TNAP+/STRO-1^(bright) 4,511 ± 185 410 TNAP+/STRO-1l^(dull) 0.0 0.0

Example 4: Relative Gene and Surface Protein Expression of STRO-1^(dull)and Stro-1^(bright) Cells

In the first series of experiments, semi-quantitative RT-PCR analysiswas employed to examine the gene expression profile of variouslineage-associated genes expressed by STRO-1^(dull) or STRO-1^(bright)populations, isolated by fluorescence activated cell sorting (FIG. 2A).In the second series of experiments, flow cytometry and mean channelfluorescence analysis was employed to examine the surface proteinxpression profile of various lineage-associated proteins expressed bySTRO-1^(dull) or STRO-1^(bright) populations, isolated by fluorescenceactivated cell sorting.

Total cellular RNA was prepared from either 2×10⁶ STRO-1^(bright) orSTRO-1^(dull) sorted primary cells, chondrocyte pellets and otherinduced cultures and lysed using RNAzolB extraction method (Biotecx Lab.Inc., Houston, Tex.), according to the manufacturer's recommendations.RNA isolated from each subpopulation was then used as a template forcDNA synthesis, prepared using a First-strand cDNA synthesis kit(Pharmacia Biotech, Uppsala, Sweden). The expression of varioustranscripts was assessed by PCR amplification, using a standard protocolas described previously (Gronthos et al., J. Bone and Min. Res.14:48-57, 1999). Primer sets used in this study are shown in Table 2.Following amplification, each reaction mixture was analysed by 1.5%agarose gel electrophoresis, and visualised by ethidium bromidestaining. RNA integrity was assessed by the expression of GAPDH.

Relative gene expression for each cell marker was assessed withreference to the expression of the house-keeping gene, GAPDH, usingImageQant software (FIG. 2B, C). In addition, dual-colour flowcytometric analysis was used to examine the protein expression profileof ex vivo expanded MPC based on their expression of a wider range ofcell lineage-associated markers in combination with the STRO-1 antibody.A summary of the general phenotype based on the gene and proteinexpression of STRO-1^(dull) and STRO-1^(bright) cultured cells ispresented in Table 3. The data indicate that ex vivo expandedSTRO-1^(bright) MPC exhibit differentially higher expression of markersassociated with perivascular cells, including angiopoietin-1, VCAM-1,SDF-1, IL-1_(β), TNFα, and RANKL. Comparisons between the protein andgene expression profiles of STRO-1^(dull) and STRO-1^(bright) culturedcells are summarised in Tables 3 and 4.

Subtractive hybridization studies were also performed in order toidentify genes uniquely expressed by STRO-1^(bright) cells. Briefly,STRO-1^(dull) and STRO-1^(bright) were isolated as described above (seeFIG. 3A). Total RNA was prepared from STRO-1^(dull) and STRO-1^(bright)cells pooled from 5 different marrow samples using the RNA STAT-60system (TEL-TEST). First-strand synthesize was performed using the SMARTcDNA synthesis kit (Clontech Laboratories). The resultantmRNA/single-stranded cDNA hybrid was amplified by long-distance PCR(Advantage 2 PCR kit; Clontech) using specific primer sites at the 3′and 5′ prime ends formed during the initial RT process according to themanufacturer's specifications. Following Rsal digestion of theSTRO-1^(bright) cDNA, 2 aliquots were used to ligate different specificadaptor oligonucleotides using the Clontech PCR-Select cDNA SubtractionKit. Two rounds of subtractive hybridization were performed usingSTRO-1^(bright) (tester) and STRO-1^(dull) (driver) cDNA, and viceversa, according to the manufacturer's protocol. This procedure was alsoperformed in reverse using STRO-1^(dull) tester cDNA hybridized againstSTRO-1^(bright) driver cDNA.

To identify genes uniquely expressed by STRO-1^(bright) population,STRO-1^(bright)-subtracted cDNA was used to construct replicatelow-density microarray filters comprising 200 randomly selectedbacterial clones transformed with the STRO-1^(bright) subtracted cDNAsligated into a T/A cloning vector. The microarrays were subsequentlyprobed with either [³²P] dCTP-labeled STRO-1^(bright) or STRO-1^(dull)subtracted cDNA (FIG. 3B-C). Differential screening identified a totalof 44 clones, which were highly differentially expressed between theSTRO-1^(dull) and STRO-1^(bright) subpopulations. DNA sequencing of allthe differentially expressed clones revealed that only 1 clone wasrepresentative of a known stromal cell mitogen; namely, platelet-derivedgrowth factor (PDGF) (Gronthos and Simmons, Blood. 85: 929-940, 1995).Interestingly, 6 of the 44 clones were found to contain DNA insertscorresponding to the chemokine, stromal-derived factor-1 (SDF-1). Thehigh abundance of SDF-1 transcripts in human STRO-1^(bright) cells wasconfirmed by semiquantitative RT-PCR of total RNA prepared from freshlysorted STRO-1^(bright), STRO-1^(dull), and STRO-1^(negative) bone marrowsubpopulations (FIG. 3D and Table 3).

TABLE 2 RT-PCR primers and conditions for thespecific amplification of human mRNA Target Product GeneSense/Antisense (5′-3′) Primer Sequences Size SEQ ID GAPDHCACTGACACGTTGGCAGTGG/ 417 SEQ ID NO: 1 CATGGAGAAGGCTGGGGCTC SEQ ID NO: 2SFD-1 GAGACCCGCGCTCGTCCGCC/ 364 SEQ ID NO: 3 GCTGGACTCCTACTGTAAGGGSEQ ID NO: 4 IL-1β AGGAAGATGCTGGTTCCCTCTC/ 151 SEQ ID NO: 5CAGTTCAGTGATCGTACAGGTGC SEQ ID NO: 6 FLT-1TCACTATGGAAGATCTGATTTCTTACAGT/ 380 SEQ ID NO: 7GGTATAAATACACATGTGCTTCTAG SEQ ID NO: 8 TNF-α TCAGATCATCTTCTCGAACC/ 361SEQ ID NO: 9 CAGATAGATGGGCTCATACC SEQ ID NO: 10 KDRTATAGATGGTGTAACCCGGA/ 450 SEQ ID NO: 11 TTTGTCACTGAGACAGCTTGGSEQ ID NO: 12 RANKL AACAGGCCTTTCAAGGAGCTG/ 538 SEQ ID NO: 13TAAGGAGGGGTTGGAGACCTCG SEQ ID NO: 14 Leptin ATGCATTGGGAACCCTGTGC/ 492SEQ ID NO: 15 GCACCCAGGGCTGAGGTCCA SEQ ID NO: 16 CBFA-1GTGGACGAGGCAAGAGTTTCA/ 632 SEQ ID NO: 17 TGGCAGGTAGGTGTGGTAGTGSEQ ID NO: 18 PPARγ2 AACTGCGGGGAAACTTGGGAGATTCTCC/ 341 SEQ ID NO: 19AATAATAAGGTGGAGATGCAGGCTCC SEQ ID NO: 20 OCN ATGAGAGCCCTCACACTCCTC/ 289SEQ ID NO: 21 CGTAGAAGCGCCGATAGGC SEQ ID NO: 22 MyoDAAGCGCCATCTCTTGAGGTA/ 270 SEQ ID NO: 23 GCGAGAAACGTGAACCTAGCSEQ ID NO: 24 SMMHC CTGGGCAACGTAGTAAAACC/ 150 SEQ ID NO: 25TATAGCTCATTGCAGCCTCG SEQ ID NO: 26 GFAP CTGTTGCCAGAGATGGAGGTT/ 370SEQ ID NO: 27 TCATCGCTCAGGAGGTCCTT SEQ ID NO: 28 NestinGGCAGCGTTGGAACAGAGGTTGGA/ 460 SEQ ID NO: 29 CTCTAAACTGGAGTGGTCAGGGCTSEQ ID NO: 30 SOX9 CTCTGCCTGTTTGGACTTTGT/ 598 SEQ ID NO: 31CCTTTGCTTGCCTTTTACCTC SEQ ID NO: 32 Collagen AGCCAGGGTTGCCAGGACCA/ 387SEQ ID NO: 33 type X TTTTCCCACTCCAGGAGGGC SEQ ID NO: 34 AggrecanCACTGTTACCGCCACTTCCC/ 184 SEQ ID NO: 35 ACCAGCGGAAGTCCCCTTCGSEQ ID NO: 36

TABLE 3 Summary of the Relative Gene Expression in STRO-1^(Bright) andSTRO-1^(Dull) populations. A list of genes which displayed measurableand differential expression between the STRO-1^(Bright) andSTRO-1^(Dull) populations as determined by reverse transcription- PCRare presented. Values represent the relative gene expression withreference to the house-keeping gene, GAPDH. Gene Expression relative toGAPDH Tissue Marker STRO-1^(Bright) STRO-1^(Dull) Neurons GFAP (Glial0.1 0.7 Fibrillary Acidic Protein) Bone OCN 1.1 2.5 (Osteocalcin) OSX(Osterix) 0.4 1.3 CBFA-1 (Core 0.3 0.6 Factor Binding Protein-1) Immuno-RANKL 1.6 0.3 regulatory (Receptor Activator of Nuclear Factor κ B)SDF-1-alpha 3.2 0.1 (Stromal Derived factor-1-alpha) Fat Leptin 3.1 4.2Cardio- GATA-4 1.1 2.9 myocytes Endothelial Ang-1 1.5 0.8 cells(Angiopoietin-1) Chondrocytes Sox 9 0.3 1.1 COL X 3.5 2.8 (Collagen X)Pro- TNF-alpha 1.7 0.9 inflammatory (Tumour necrosis Cytokines alpha)

To correlate protein surface expression with density of STRO-1expression, single cell suspensions of ex vivo expanded cells derivedbone marrow MPC were prepared by trypsin/EDTA detachment andsubsequently incubated with the STRO-1 antibody in combination withantibodies identifying a wide range of cell lineage-associated markers.STRO-1 was identified using a goat anti-murine IgM-fluoresceinisothiocyanate while all other markers were identified using either agoat anti-mouse or anti-rabbit IgG-phycoerythrin. For those antibodiesidentifying intracellular antigens, cell preparations were firstlabelled with the STRO-1 antibody, fixed with cold 70% ethanol topermeabilize the cellular membrane and then incubated with intracellularantigen-specific antibodies. Isotype matched control antibodies wereused under identical conditions. Dual-colour flow cytometric analysiswas performed using a COULTER EPICS flow cytometer and list mode datacollected. The dot plots represent 5,000 listmode events indicating thelevel of fluorescence intensity for each lineage cell marker (y-axis)and STRO-1 (x-axis). The vertical and horizontal quadrants wereestablished with reference to the isotype matched negative controlantibodies.

TABLE 4 Summary of the Relative Protein Expression in STRO-1^(Bright)and STRO-1^(Dull) populations. A list of proteins which displayeddifferential expression between the STRO-1^(Bright) and STRO-1^(Dull)populations as determined by flow cytometry are presented. Valuesrepresent the relative mean fluorescence intensity of staining. MeanFluorescence Intensity Tissue Marker STRO-1^(Bright) STRO-1^(Dull)Neurons Neurofilament 1.7 20.5 Bone ALK PHOS 5.7 44.5 (AlkalinePhophatase) Immuno- RANKL (Receptor 658.5 31.0 regulatory Activator ofNuclear Factor κ B) Epithelial Cells CytoKeratin 1.2 23.3 10 + 13Cytokeratin 14 1.8 8.8 Smooth Muscle α-SMA (Alpha 318.0 286.0 SmoothMuscle Actin ) Chondrocytes Byglycan 84.4 65.9 Basal Fibroblast TenascinC 22.2 6.9 Cardiomyocyte Troponin C 2.5 15.0

These results show that SDF-1alpha and RANKL are highly expressed bySTRO-1^(bright) cells. This is important because both of these proteinsare known to be involved in up-regulation of CD4+ CD25+ regulatory Tcells which confer protection against immune disorders such as GVHD(Loser et al., Nature Medicine 12:1372-1379, 2006; Hess, Biol. BloodMarrow Transplant, 12 (1 Suppl 2):13-21, 2006; and Meiron et al., J.Exp. Medicine 205:2643-2655, 2008).

Example 5: In Vitro Immunosuppressive Activity

To assess immunosuppressive activity of culture-expanded STRO-1^(bright)cells (MPC(B)), we used CD3/CD28 stimulation as a read-out. Results werecompared to a population of culture-expanded, bone marrow-derived STRO-1negative cells isolated as in Example 1 (MSC(A)). Human peripheral bloodmononuclear cells (PBMC) were stimulated with CD3/CD28 coated beads inthe presence of 4 escalating concentrations of MSC and MPC preparations.The proliferation of T cells was measured by 3H-Tdr incorporation.

MSC (A) and STRO-1^(bright) MPCs (B) were tested for their ability tosuppress the response of human peripheral blood mononuclear cells (PBMC)to CD3/CD28 stimulation. MSC and MPC or commercially-purchased controlhuman MSC (Lonza) were added at different ratios to the cultures ofPBMC. After 3 days, 3H-Tdr was added for 18 hours and the cultures thenharvested.

PBMC proliferation in response to CD3/CD28 was inhibited in a dosedependent fashion by all preparations. However, preparation B wasclearly superior to the effect produced by preparation A as well ascontrol hMSC (FIG. 4). At a 1:100 MSC:PBMC ratio, MPC B still inhibited70% of control T cell proliferation, whilst controlcommercially-purchased MSC (Lonza) and MSC A produced a 50% and 60%inhibition, respectively (FIG. 5).

Example 6: Induction and Treatment of GvHD

The in vivo immunosuppressive activity of STRO-1^(bright) cells (MPC(B))was investigated using a model of graft-versus-host disease (GvHD) basedon a donor recipient pair mismatched for multiple minorhistocompatibility loci. T-cell depleted bone marrow mononuclear cells(BMMC) (5×10⁶) and splenocytes (30×10⁶) from B10.D2 (H2d) donors wereinjected intravenously into lethally irradiated (750 cGy) BALB/c (H2d)recipient mice. In this situation the splenic lymphocytes from B10.D2recognise and attack BALB/c recipient tissues and produce weight loss,fibrosis and hair loss. The disease was monitored using the conventionalscoring system by weighing the animals and assessing skin manifestationsfrom week 4-5 after the transplant. As a comparison, immunosuppressiveactivity of bone marrow-derived STRO-1 negative cells isolated as inExample 1 (MSC(A)) was evaluated.

Whereas a positive control group of mice did not receive any furthertreatment, the experimental groups were injected intravenously with MSCs(A) or STRO-1^(bright) MPCs (B) at a dose of 2×10⁶, 1×10⁶, or0.3×10⁶/mouse from week 4 every week for 3 times. Mice were monitoredtwice a week. Each group contained eight mice.

Example 7: Effects of MSCs and MPCs on the Development of GvHD

Mice received 1 or 2×10⁶ MSC A (A1 and A2), 1 or 2×10⁶ MPC B (B1 andB2). The kinetics of disease in the absence or presence of MSC treatmentare reported in FIG. 6. Following the infusion of 1×10⁶ cells, there wasa clear difference between the effects of B and A. Whilst mice receivingpreparation A did not exhibit any substantial difference from thosereceiving no cells, the group injected with preparation B showed adramatic beneficial effect on the severity of the disease. Thirteenweeks after the transplant, mice which had received B1 had an averageGvHD score of 0.5 as compared to 2.3 in the other groups.

We then investigated whether the anti-GvHD effect was dose dependent.Therefore a group of mice was injected with a higher dose (2×10⁶ permouse) and one with a lower dose (0.3×10⁶ per mouse) of A or B accordingto the same modalities described for the previous dose. FIG. 7 reportsthe effects of the highest dose. The therapeutic effect of high dose MPC(B2) was superior relative to A2, with no GvHD seen at all for the first11 weeks in this group. At 14 and 15 weeks, the consequences of GvHD permouse was even more dramatic (FIG. 8) with no mice at all surviving inthe A1 group.

Lastly, the injection of a lower dose (0.3×10⁶ per mouse) againdemonstrated that by 9 weeks STRO-1^(bright) MPCs (B) had a superioreffect on GVHD score reduction than A (FIGS. 9 and 10).

The data of this pilot study have consistently shown thatSTRO-1^(bright) MPCs exhibited superior immunosuppressive capacities ascompared to either no treatment or treatment with STRO-1 negative MSCs.This was evident in the in vitro assay and, most importantly in the invivo assay. STRO-1^(bright) MPCs produced a dramatic clinical effect onthe prevention of GvHD given at dose ranges of 0.3-2×10⁶ cells permouse.

All references cited in this document are incorporated herein byreference.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The invention claimed is:
 1. A method for preventing the development ofor treating graft versus host disease (GvHD) complications in a humanpatient undergoing or about to undergo a bone marrow or hematopoieticstem cell transplantation, which method comprises administering, to thehuman patient, a therapeutically effective amount of a population ofcells enriched for STRO-1⁺, TNAP⁺ mesenchymal precursor cells (MPCs), ormultipotential cells culture-expanded from STRO-1⁺, TNAP⁺ MPCs, whereinthe cells in the administered population are CD34⁻.
 2. The methodaccording to claim 1, wherein the administered population isadministered to the human patient prior to undergoing thetransplantation.
 3. The method according to claim 1, wherein the bonemarrow or hematopoietic stem cell transplantation is an allogeneictransplantation.
 4. The method according to claim 1, wherein theadministered population is allogeneic.
 5. The methods according to claim1, wherein the population is administered systemically.
 6. The methodaccording to claim 5, wherein the population is administered byintravenous injection.
 7. The method of claim 1, wherein theadministered population comprises between 0.1×10⁶ to 5×10⁶ cells.
 8. Themethod of claim 1, wherein the administered population comprises between0.3×10⁶ to 2×10⁶ cells.
 9. The method of claim 1, wherein theadministered population comprises between 0.1×10⁵ and 0.5×10⁶ cells. 10.The method of claim 9, wherein the administered population comprisesabout 0.3×10⁶ cells.
 11. The method of claim 1, wherein the administeredpopulation is administered once weekly or less often.
 12. The methodaccording to claim 1, wherein the human patient is suffering fromaplastic anemia, myelofibrosis, or bone marrow failure followingchemotherapy and radiation therapy.
 13. The method according to claim 1,further comprising administering an immunosuppressive drug to the humanpatient.